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Biochemical and biological characterization of exosomes containing prominin-1/CD133

机译：含有prominin-1 / CD133的外泌体的生化和生物学特性

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Exosomes can be viewed as complex “messages” packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic- microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.
机译：外泌体可以看作是复杂的“信息”，被包装成可以存活到局部微环境中其他细胞的行程，并通过体液到达远处。大量证据表明某些类型的癌症外泌体具有促转移作用。我们以前曾报道过prominin-1在黑色素瘤细胞中具有促转移作用，而从转移性黑色素瘤细胞释放的微泡表达了高水平的prominin-1。为了探索控制癌症外泌体中蛋白-脂质-微RNA分选的机制及其对转移表型的潜在贡献，我们在此采用基于prominin-1的免疫磁分离技术结合过滤和超速离心来纯化prominin-黑色素瘤和结肠癌细胞的1表达外泌体（prom1-exo）。 Prom1-exo包含154种蛋白质，包括在外泌体中最频繁表达的所有14种蛋白质，以及多种前转移性蛋白质，包括CD44，MAPK4K，GTP结合蛋白，ADAM10和Annexin A2。它们的脂质组成类似于筏的微区，富含溶血磷脂酰胆碱，溶血磷脂酰乙醇胺和鞘磷脂。大量的四跨膜蛋白和四跨膜蛋白相关蛋白，以及高水平的鞘磷脂，表明脂蛋白组装体，可能是四跨膜蛋白网，可能是干细胞和癌症干细胞中prominin-1释放过程中的重要结构决定因素。 Micro-RNA分析显示，prom1-exo中49种micro-RNA的浓度高于亲本细胞，其中20种具有癌症相关功能。将prom1-exo添加到黑色素瘤和骨髓源性基质细胞（MSC）培养物中3小时后，观察到大量积累。黑色素瘤细胞和MSC的短期共培养导致异源prominin-1转移。 MSC暴露于prom1-exo可增加其侵袭性。我们的研究支持这样的概念，即特定的癌症外泌体群体包含多个决定其来源的细胞转移潜能的决定因素。

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